Derivatives of 3beta-hydroxy steroids and the 6- and 16-substituted analogs thereof



United States Patent 3,209,000 OF 3,6-HYDROXY STEROIDS AND16-SUBSTITUTED ANA THEREOF DERIVATIVES AND THE 6- LOGS John o. Babcockand J Allan Campbell, Kalamazoo,

Mich., assignors to The Upjohn Company, Kalamazoo, 'Mich., a corporationof Delaware No Drawing. Filed May 1, 1963, Ser. No. 277,089

21 Claims. (Cl. 260239.5)

10 This invention relates to novel steroids and more pa ticularly to thederivatives of 3fi-h'ydroxy steroids and methods used in the preparationthereof.

Some of the novel compounds of this invention can be represented by I(a) Those having the formulae CH3 CH3 CH3 CH3 =0 7 0 -"OAc 0H3 -OAc v pCH3 CH3 (en CH; 0H 1 0H 3 --OAc I 3 -OAc p: IICHZ FCH; a

10 RlO- 40 iii X1 CH3 CH3 CH CH on =o 0H a 0 Ac 3 OAc A j j a R1O- R10-wherein Ac is the acetyl radical, X is selected firom the groupconsisting of hydrogen, methyl, fluoro, chloro, trifiuoromethyl,difluorornethyl, and fluoromethyl, X is selected from the groupconsisting of hydrogen and methyl, R is selected from the groupconsisting of ice and

and vR is selected from the group consisting of '(i) The acyl radical ofa dib asic organic carboxylic acid, particularly a hydrocarbondioarboxylic acid, containing from 3 to 12 carbon atoms, inclusive,

(ii) An aminoacyl radical of an amine substituted hydrocarbon canboxylicacid, such as an aminosubstituted acetyl radical of the formula whereinR is selected from the group consisting of the .pyrrolidino, piperidinoand morpholino radicals and a radical of the formula wherein R and R areselected from the group consisting of the benzyl radical and lower"alkyl radicals containing from 1 to 4 carbon atoms, inclusive;

'(b) The alkali metal salts of (a) (i); and

(c) The pharmacologically acceptable acid addition and quaternaryammonium salts of (a) (ii).

The compounds of (a), (b) and (c) above are effective antifertility,estrus regulating, progestational, and pregnancy maintaining agents ofimproved therapeutic ratio and are particularly useful in the treatmentof mammals. The compounds above are also useful in the treatment ofinflammatory conditions of mammals and birds and are particularly usefulin the treatment of inflammatory conditions of the skin, eyes and earsof humans and of valuable domestic animals, as well as contactdermatitis, lan aphylaxis, and other allergic reactions,

Administration of the novel steroids of this invention can be inconventional dosage forms, such as pills, tablets, capsules, syrups orelixirs for oral use, or in liquid forms which are adaptable to thenatural and synthetic cortical hormones for injecta-ble products; theycan also be administered topically in the form of ointments, creams,lotions, and the like, with or without coating antibiotics, germicidesor other materials forming advantageous combinations therewith.

In addition, the compounds of (a), (b) and (c), particularly thecompounds of (b) and (c), can be dissolved to a useful extent in aqueousvehicles to provide soluble formulations which are useful forintravenous and intraamniotic administration in treating habitual orthreatened abortion. These aqueous solutions are also useful for oraladministration (and may be formed in situ by injection of thewater-soluble drug in the solid state) since they provide essentiallyimproved elficacy and potency,

5 rapid onset of activity, anda prolonged effect.

Q HO- J Esterification with di- Haloacetic acid carboxylic acids oresterification derivatives thereof \1 C H3 H3 Q N Q rno- J zo H,CO- J(II) (IV) Amine Salt formation formation 0 H Direct (III) Alkaliquatermetal salts x nary of the oom- O Q ammonounds of (U) J iurnhaormula (II) Rz-CHr 0- lide formation Acid addition Quaternary saltformation ammonium salt format- ,Lion

(VI) Acid addition salt (VII) Quaternary ammoncorrespondiug to ium saltcorrespond- Formula V ing to Formula V wherein Q is the remainder of thesteroid moiety having B-, C- and D-ring configurations selected from thegroup consisting of CHa i TCH; TCHQ in which Ac, X, X and R are definedas above, R is defined as above, R is the acyl radical of a hydrocarbondicarboxylic acid containing from 3 to 12 carbon atoms, inclusive, and Zis selected from the group consisting of chlorine and bromine.

It is to be understood that the discussion which follows is not to beconstrued as being limited to the specific compounds named but isapplicable to, and embracive of, all the compounds disclosed within thepurview of the flow sheet.

The novel compounds of the present invention are prepared by thefollowing reactions:

ESTERIFICATION WI'I I-I HY DROCARBON DICAR- BOXYIJIC ACIDS ORDERIVATIVES THEREOF The compounds of Formula I can be converted to their3B-acylates, the compounds of Formula II, by methods known in the art.This is accomplished by allowing the compounds of Formula I, such as35,17a-di'hydroxy-4- pregnen-20-one 17-aoetate, to react with anacylating agent thus yielding the compounds of Formula H, such as 35,17a-dihydroxy-4-pregnen-Q0-one 3-acyla-te 17-acetate.

Suitable acyla-ting agents are dibasic organic carboxylic acids,particularly hydrocarbon dicarboxylic acids, containing from 3 to 12carbon atoms, inclusive, or the anhydrides or acid halides thereof. .Forexample, a saturated dibasic acid, e.g., malonic succinic, glutaric,adipic, pimelic, suberic, ufi-dimethylsuccinic, a,;8-diethylsuccinic,fi-methylglutanic, a,p-dimethylglutaric, a,a-diethylglutaric,fi,fi-dimethylglutaric, aethyl-Bmethylglutaric,fi-ethyl-fimethylglutaric, a,a,p-trimethylglutaric,afifi-trimethylglutaric, a,;9,' -trimethylglutaric,u-ethyl-fi,fi-dimethylglutaric, a,a,5,o-tetramethylglutaric,a,;3,fi,'y-I6tIlam6thylgllJ- tal'ic, p-methyl-p-butylglutaric,fi-methyl-fi-is-obutylglutaric, u-methyladipic, a,a-dirnethyladipic, andthe like; dibasic unsaturated acids, e.g., maleic, citraconic; 0-, rn-,pphthalic acid; diglyco'lic acid, and the like; or the acid anhydridesand acid halides thereof, can be used to acylate the compounds ofFormula I to convert them into the compounds of Formula II.

The conversion of the compounds of Formula I to the compounds of FormulaII is carried out using the acylating agent as solvent, or preferably inthe presence of a suitable inert solvent, such as benzene, xylene,dioxane, methylene chloride, ether, and the like, particularly when theacylating agent is a solid, and frequently in the presence of acatalyst, such as p-toluenesulfonic acid, or an amine, preferablypyridine. Completion of the reaction can take from a few minutes to 24hours, depending on the temperature and the solvent employed. If theacylating agent is the free acid, the reaction is preferably carried outin the presence of an esterification catalyst, for example,p-toluwesulfonyl chloride, trifluoroacetic anhydride, p-toluenesulfonicacid, trifluoroacetic acid, sulfuric acid, and the like.

The thus obtained compounds of Formula II are recovered from thereaction mixture by conventional methods, such as, for example,precipitation by dilution with water or dilute inorganic acid andcollection of the 3- acylated reaction production, such as3p,17a-dihydroxy- 4-pregnen-20-one 3-hemisuccinate 17-acetate, byfiltration or extraction with a water-immiscible solvent such asmethylene chloride, ethyl acetate, benzene, ether, and the like, directcrystallization, chromatography, counter-current extraction or acombination of these, followed by final crystallization if desired.

The S-acylate 17-acetate steroids of Formula II can, if desired, be usedin the salt formation step in the crude state.

SALT FORMATION (II)- (III) The compounds of Formula II, such as, forexample, 3,3,17a-dihydroxy-4-pregnen-20-one S-hemisuccinate 17- acetate,are converted to water-soluble salts, the alkali metal salts, i.e.,sodium, potassium or lithiumsalts, the compounds designated III, such as3,6',l7a-dihydroxy-4- pregnen-20-one 3-sodium snccinate 17-acetate,according to procedures well known in the art for the preparation ofwater-soluble salts, e.g., the preparation of water solublesalts ofhydrocortisone hemiacylates from hydrocortisone ZI-hemiacylates,disclosed in US. Patent 3,025,311.

HALOACETIC ACID ESTERIFICATION (I) (IV) Reaction of the compounds ofFormula I, such as, for example, 3p,17a-dihydroxy-4-pregnen-20-one17-acetate, with bromoacetic anhydride (or bromoacetyl chloride) orchloroacetic anhydride (or chloroacetyl chloride) gives thecorresponding 3-bromoacetate or 3-chloroacetate ester respectively ofthe compounds of Formula IV, such as, for example,3B,17a-dihydroxy-4-pregnen-20-one 3-bromoacetate l7-acetate or35-17a-dihydroxy-4-pregnen-20-one 3-chloroacetate 17-acetate.

The 3-haloacetic esterification is preferably carried out using asolution of the haloacetic anhydride in an inert solvent, such as ether,tetrahydrofuran, methylene, chloride, benzene, and the like, preferablyin the presence of pyridine as catalyst, as known in the art. Thereaction is carried out at temperatures from about 0 to 60 C., withtemperatures of from about 0 to 30 C. being preferred. Preferably thereaction is also carried out under an inert atmosphere, e.g., nitrogen.

The thus obtained compounds of Formula IV are recovered from thereaction mixture and purified by conventional methods, such as, forexample, those described above for the recovery and purification of thecompounds of Formula II. However, the compounds of Formula IV need notbe purified to a high degree since the crude reaction product containingthem can be used directly in the next succeeding step, the amineformation.

AMINE FORMATION (IV) (V) The compounds of Formula IV, such as, forexample, 3B,17a-dihydroxy-4-pregnen-20-one 3-bromoacetate 17- acetate or3,8,17ot-dihydroxy-4-pregnen-20-one 3-chloro acetate 17-acetate, arereacted with more than one equivalent, and preferably with at least twoequivalents, of the selected secondary amine to yield the corresponding3- aminoacetate esters, the compounds of Formula V, such as, forexample, 35,17wdihydroxy-4-pregnen-20-one 3-dimethylaminoacetate17-acetate, according to procedures known in the art.

Suitable secondary amines are, for example, dimethylamine, diethylamine,dipropylamine, dioctylamine, methylethylamine, methylpropylamine,dibenzylamine, methylbenzylamine, pyrrolidine, piperidine, morpholine,and the like.

The reaction is carried out in an inert solvent, e.g., benzene, toluene,ether, dioxane, tetrahydrofuran, commercial hexanes, carbontetrachloride, methylene chloride, 2-butanone, and the like, attemperatures from about C. to the reflux temperature of the reactionmixture, with to 60 C. being preferred, for a period of from 1 to 48hours. The reaction mixture is filtered and the compounds of Formula V,such as, for example, 3B,17a-dihydroxy-4-pregnen-20-one3-dimethylaminoacetate 17- acetate, are recovered from the filtrate andpurified by conventional methods, such as, for example, those describedabove for the recovery and purification of the compounds of Formula II.However, the compounds of Formula V need not be purified to a highdegree since they can be used in the following steps, the acid additionor quaternary ammonium salt formation, in the crude state.

ACID ADDITION SALT FORMATION (V) (VI) The compounds of Formula V, suchas, for example, 3/3,17a-dihydroxy-4-pregnen-20-one3-dimethylaminoacetate 17-acetate, are reacted with a pharmacologicallyacceptable acid, such as, for example, formic, acetic, propionic,hydrochloric, hydrobromic, hydriodic, sulfuric, phosphoric, nitric,lactic, pamoic, and the like, to produce 6 the acid addition salts ofthe compounds of Formula V, the compounds designated VI, such as, forexample, 35, 17a-dihydroxy-4-pregnen-20-one 3-dimethylaminoacetate17-acetate hydrochloride.

The 3-aminoacetate ester is treated in an inert solvent with theappropriate acid and recovered under conditions well known in the artfor the preparation and recovery of amine acid addition salts.

In a preferred procedure a solution containing a slight excess ofpharmacologically acceptable acid in an inert solvent, such as, forexample, ether, Z-butanone, dioxane, tetrahydrofuran, and the like, isadded slowly to a well stirred solution of the 3-aminoacetate ester. Theacid addition salt precipitates and is collected by filtration. Ifdesired, the thus produced acid addition salt can be further purified byrecrystallization.

QUATERNARY AMMONIUM SALT FORMATION (V1I) The compounds of Formula V,such as, for example, 3 8,17a-dihydroxy-4-pregnen-20-one3-dimethylaminoacetate 17-acetate, are reacted with a lower-alkylhalide, such as, for example, a lower-alkyl bromide, iodide or chloride,preferably methyl or ethyl bromide, and the like, to produce thequaternary ammonium compounds of Formula V, designated as VII, such as,for example, 3B,17a-dihydroxy-4-pregnen-20-one 3-dimethylaminoacetate17-acetate methobromide.

The 3-aminoacetate ester is treated with the appropriate lower-alkylhalide and recovered, under conditions Well known in the art for thepreparation and recovery of quaternary ammonium salts.

In a preferred procedure a solution of the alkyl halide in ether isadded to a solution of the 3-aminoacetate ester in an inert solvent,such as, for example, benzene, ether, dioxane, Z-butanone, and the like,and stirred at a temperature of from about 10 to C. for about 1 tohours. The quaternary ammonium salt which precipitates is collected byfiltration and, if desired, purified by recrystallization.

DIRECT QUATERNARY AMMONIUM HALIDE FORMATION (IV)- (VII) Alternatively,the compounds of Formula IV, such as, for example,3B,17wdihydroxy-4-pregnen-20-one 3-bromoacetate 17-acetate or318,l7u-dihydroxy-4-pregnen-20- one 3-chloroacetate l7-acetate, arereacted with one or more equivalents of a tertiary amine, such as, forexample, trimethylamine, triethylamine, tripropylamine,dimethylethylamine, benzyldimethylamine, pyridine, and the like, toyield the corresponding 3-aminoacetate quaternary ammonium halides, thecompounds embraced by designation VII, such as, for example,3B,17a-dihydroxy-4-pregnen-20-one 3-dimethylaminoacetate 17-acetatemethobromide or 36,17a-dihydroxy-4-pregnen-20-one S-dimethylaminoacetate17-acetate methochloride.

The reaction is carried out in an inert solvent, such as, for example,benzene, toluene, ether, dioxane, tetrahydrofuran, commercial hexanes,carbon tetrachloride, methylene chloride, 2-butanone, and the like, attemperatures of from about 0 to 100 0., preferably at about 20 to 60 C.,for 1 to 48 hours. The 3-aminoacetate quaternary ammonium halide whichprecipitates is collected by filtration and, if desired, purified byrecrystallization.

The following preparations and examples illustrate the best modecontemplated by the inventors for carrying out their invention, but arenot to be construed as limiting.

PREPARATION 1 6 a-difluoromethyl-J 7 u-hydroxy-4-pregnene-3,20-di0ne 1 7acetate A. 6a-HYDROXYMETHYL-Bfi,17a-DIHYDROXY-5a- PREGNAN-Z O-ONE3,17-DIACETATE A mixture of g. of the known compound 3,8,1741-dihydroxy-5-pregnen-20-one 3,17-diacetate, 1875 ml. of

toluene and 18.75 g. of cobalt carbonate catalyst was placed in a 1gallon stainless steel autoclave equipped with a mechanical stirrer. Thevessel was flushed three times with carbon monoxide and the pressuretherein brought to 450 p.s.i. with carbon monoxide, then to 1150 p.s.i.,with hydrogen and heated at 180 C. for a period of about 18 hours. Thereaction mixture was cooled and filtered through a bed of Celite(diatomaceous earth) and the clear yellow filtrate concentrated todryness on a rotary evaporator. The residue was triturated with amixture of ethyl acetate and ether and the crystals collected and washedwith ether to yield 71.2 g. of product with a melting point of 225 to231 C. An additional 4.6 g. was obtained from the mother liquor. Aportion of the product was recrystallized from ethyl acetate .to yieldan analytical sample of6a-hydroxymethyl-3fi,17a-dihydroxy-5a-pregnan-20-one 3,17-diacetatehaving a melting point of 232 to 234 C. and a rotation [a] of -1(chloroform).

Analysis.-Calcd. for C H O C, 69.01; H, 8.99. Found: C, 69.64; H, 8.72.

B. 6a p TOLUENESULFONOXYMETHYL 35,170. DIHY- DROXY-5a-PREGNAN20'ONE3,17-DIACETATE 14.3 g. of p-toluenesulfonyl chloride was added to asolution of 14.3 g. of6a-hydroxymethyl-3fi,17u-dihydroxy-5u-pregnan-20-one 3,17-diacetate in100 ml. of pyridine. The solution was kept in a cold bath for a fewminutes, then at room temperature for a period of about 18 hours. Thesolution was then poured into dilute sodium bicarbonate solution and theresulting precipitate collected on a filter, thoroughly washed withwater and dried to yield 17.8 g. of product having a melting point of197 to 208 C. Two recrystallizations from ethyl acetate gave ananalytical sample of6a-ptoluenesulfonoxymethyl-3fi,l7a-dihydroxy-5u-pregnan-ZO- one3,17-diacetate, melting at 214 to 216 C. with rotation [a1 (chloroform)of +1".

Analysis.Calcd. for C H O S: C, 65.28; H, 7.53; S, 5.45. Found: C,65.82; H, 7.82; S, 5.23.

C. 6a-FORMYL-3B,1Ta-DIHYDROXY-Sa-PREGNAN- 20-ONE 3,17-DIACETATE Asuspension of 20 g. of sodium bicarbonate in 125 ml. ofdimethylsulfoxide with nitrogen bubbling therethrough was heated in anoil bath held at a temperature of about 155 C. When the temperature ofthe suspension reached 135 C., 12.3 g. of6a-p-toluenesulfonoxymethyl-3,8,17adihydroxy-Saregnan-ZO-one3,17-diacetate was added thereto. The flask containing the suspensionWas swirled intermittently for a period of about 10 minutes. Thereaction mixture was cooled to about 40 C., then poured into ice-water.The amorphous precipitate was collected, washed with water and dried toyield 9.05 g. of product. A 1 g. aliquot of this material waschromatographed through a 100 g. Florisil (synthetic magnesium silicate)column using gradient elution, employing 3 l. of a mixture ofSkellysolve B (hexane hydrocarbons) containing 4% acetone and 3 l. of amixture of Skellysolve B containing 12% of acetone and collecting 250ml. fractions to yield the desired aldehyde (fractions 13 to 17). Theproduct obtained from these eluates was recrystallized twice from etherto give 0.2 g. of dense crystals of6aformyl-3B,17a-dihydroxy-5a-pregnan-ZO one 3,17-diacetate with amelting point of 170 to 172 C. (decomposition) and rotation [041(chloroform) of +28".

Analysis.Calcd. for C H O C, 69.92; H, 8.58. Found: C, 70.01; H, 8.70.

D. 6a-DIFLUOROMETHYL-3fl,17a-DIHYDROXY-5a- PREGNAN-ZO-ONE 3,17-DIACETATE2 g. of unpurified 6a-formyl-3B,17a-dihydroxy-5a-pregnan-20-one3,17-diacetate in 20 ml. of methylene chloride and 0.2 ml. of water wastreated with 40 g. of sulfur tetrafiuoride in a stainless steel rockingautoclave at room temperature for a period of about 16 hours. Thereaction mixture was washed with sodium bicarbonate solution and water,dried and the solvent removed. The residue was dissolved in methylenechloride and chromatographed through a g. Florisil column packed wetwith Skellysolve B and eluted by gradient elution with 4 l. of a mixtureof 3% acetone in Skellysolve B and 4 l. of a mixture of 12% acetone inSkellysolve B taking 250 ml. fractions. The desired difluoromethylcompound was eluted in fractions 14 to 21. The residues of thesefractions were combined and recrystallized from a mixture of acetone andSkellysolve B to yield 1.15 g. of this material. Recrystallization fromthe same solvents gave pure lightcolored 6u-difiuoromethyl-3[3,17u-dihydroxy-5a-pregnan- 20-one 3,17-diacetate melting at 154 .to156 C.

Analysis.Calcd. for C H F O C, 66.64; H, 8.17. Found: C, 67.04; H, 8.17.

E. Ga-DIFLUOROMETHYL-3fl.17a-DIHYDROXY-5a- PREGNAN-20-ONE 17-ACETAIE Asolution of 1.9 g. of6a-difiuoromethyl-3B,17a-dihydroxy-5a-pregnan-20-one 3,17-diacetate washeated at refluxing temperature with 40 ml. of methanol and 0.4 ml. ofconcentrated hydrochloric acid for a period of about 1 hour. Water wasadded and most of the methanol evaporated with a fast stream ofnitrogen. The resulting precipitate was collected by filtration, washedwith water and dried to yield 1.6 g. of product. A small amount of thismaterial was recrystallized from a mixture of acetone and Skellysolve Bto yield Ga-difiuoromethyl- 3,8,17x-dihydroxy-5a-pregnan-20-one17-acetate with a melting point of 197 to 205 C. and a rotation [04],;(chloroform) of +12.

Analysis.-Calcd. for C H F O C, 67.57; H, 8.51. Found: C, 67.54; H,8.61.

F. Ga-DIFLUOROMETHYL-l7a-HYDROXY-5a- PREGNANEBQO-DIONE 17-ACETATE 0.9 g.of 6a-difluoromethyl-3B,17a-dihydroxy-5a-pregnan-20-one 17-acetate wasadded to a solution of 0.9 g. of sodium dichromate dihydrate in 9 ml. ofacetic acid. The solution was stirred for a period of about 5 hours andpoured into water. The resulting precipitate was collected byfiltration, washed with water and dried. It was chromatographed througha 75 g. column of Florisil previously packed wet with Skellysolve B;gradient elution of the product was carried out with mixtures of 3 l. of2% acetone in Skellysolve B and 3 l. of 12% acetone in Skellysolve Btaking 250 ml. fractions. Fractions 22 to 27 gave 0.6 g. of the 3-ketonemelting at 188 to 192 C. after recrystallization from acetone. A smallportion of this material was again recrystallized from acetone to yieldpure light-colored 6a-difiuoromethyl-17a-hydroxy- 5a-pregnane-3,20-dione17-acetate having a melting point of 192 to 193 C. and a rotation [4x1(chloroform) of +17.

Analysis.Calcd. for C H F O C, 67.90; H, 8.07. Found: C, 68.10; H, 8.34.

G. 6a-DIFLUOROMETHYL-17a-HYDROXY-4- PREGNENE-3,20DIONE 17-ACETATE 410mg. of 6a difluoromethyl 17a hydroxy-Sa-pregnane-3,20-dione 17-acetatein 10 ml. of dioxane was acidified with a drop of 4 N hydrogen bromidein dioxane and 320 mg. of bromine added over a period of 1 minute. Aftera period of about 1 hour at room temperature, an excess of aqueoussodium bicarbonate solution was added to the reaction mixture. Theprecipitated 2,4-dibromo derivative ofGa-(lifiHOIOn'lBthYl-I7a-hYdIOXY-5oc-PI6gI18I1- 3,20-dione 17-acetatewas treated with 0.9 g. of sodium iodide in 15 ml. of acetone containingbromoacetone, and the mixture heated at refluxing temperature for aperiod of about 2.5 hours. 0.3 g. of oxalic acid was then added andheating continued for a period of about 1 hour. After cooling, ethylacetate was added and the solution filtered. The filtrate was washedwith water and sodium bicarbonate solution, then dried with sodiumsulfate. The filtrate was stirred with 500 mg. of zinc dust in 2 ml. ofacetic acid for about 1 hour and then filtered. The organic layer waswashed successively with water, sodium 9 bicarbonate solution and driedwith sodium sulfate. Evaporation of the solvent gave the crudeu,/3-unsaturated ketone, which on purification with a Girard reagent,followed by subsequent crystallization yielded pure lightcolored 6adifluoromethyl-17a-hydroxy-4-pregnene-3,20- dione 17-acetate.

Alternatively, if desired, the crude a,fl-unsaturated ketone can bepurified by chromatography over Florisil with increasing proportions ofacetone in Skellysolve B, followed by recrystallization.

H. 6a-DIFLUOROMETHYL 17a-HYDROXY-4-PREGNENE 3,20-DIONE 17-ACETATE Amedium consisting of 1% dextrose hydrate, 2% cornsteep liquor of 60%solids and tap water was adjusted to pH 4.9 with sodium hydroxide. Themedium was steam sterilized at 15 pounds pressure for about 30 minutes,cooled and then inoculated with a 24 hour growth, from spores, ofNocardia blackwellii (NCTC 630 [Medical Research Council of the ListerInstitute, London]). The medium was agitated and sparged with sterileair at the rate of one-tenth volume of air per volume of medium perminute. At the end of 24 hours of fermentation at room temperature, thepH was about 7.4. To this culture there was added a solution of6a-difluoromethyl-l7u-hydroxy-5ot-pregnane-3,20-dione 17-acetatedissolved in a minimal amount of dimethylformamide. The solution wasprepared by dissolving 5 parts of the steroid in 100 parts of thesolvent and adding about ml. of the solution per liter of the medium.Fermentation was continued for a period of about 6 hours whereupon themycelium and beer were extracted thoroughly with methylene chloride. Theextract was washed with sodium bicarbonate solution and then with water,dried and concentrated under vacuum to give6u-difiuoromethyl-l7a-hydroxy-4-pregnene-3,20 dione.

Instead of Nocardia blackwellii (NCTC 630 [Medical Research Council ofthe Lister Institute, London]) to produce fermentative dehydrogenationat the 4,5 position, other microorganisms may be similarly effectivelyemployed; included are those chosen from the group consisting of: ATCC4275 (Nocardia convoluta); ATCC 9604 (Norcardia gardneri) and NRRLB-1365 (Nocardia coelz'aca).

A solution composed of 1 g. of6oz-difluoromethyl-17ahydroxy-4-pregnene-3,20-dione, 2.5 ml. ofdistilled acetic anhydride, 250 mg. of p-toluenesulfonic acid and 2.5ml. of acetic acid was stirred for a period of about 90 minutes. Themixture was poured with vigorous stirring into water. The precipitatedsolid was separated by filtration, dried, chromatographed over Florisilwith increasing proportions of acetone in Skellysolve B andrecrystallized from ethyl acetate to yield light-colored6a-difluoromethyl-l7a-hydroxy-4-pregnene-3,20-dione l7-acetate.

PREPARATION 2 6-diflu0r0methyl-1 7a-hydr0xy-4,6-pregnadiene-3,20-di0ne J7 -acetate 1 g. of 6a difluoromethyl 17a hydroxy-4-pregnene- 3,20-dione17-acetate, 1.5 g. of recrystallized2,3,5,6-tetrachloro-l,2-benzoquinone (chloranil) and 60 ml. of tertiaryamyl alcohol were heated to boiling under nitrogen with a few boilingchips, and gently refluxed for a period of about 6 hours. The mixturewas cooled and evaporated to dryness under reduced pressure. The solidresidue (with the exception of some chloranil, which was insoluble) wasdissolved in about 100 ml. of ether and filtered. The chloranil on thefilter paper was washed with several portions of ether and the combinedether filtrates washed with 200 ml. portions of cold 2% sodiumhydroxide. The ether filtrates were washed with cold water until thewashings were neutral, then with saturated sodium chloride solution. Thepooled ether solutions were dried over sodium sulfate and evaporated todryness. This residue dissolved in methylene chloride waschromatographed over a g. column of Florisil and eluted with fractionsof Skellysolve B containing increasing proportions of acetone. The lastthird of the fractions were evaporated to dryness. The residue wasrecrystallized twice from cold methanol to yield pure light-coloredcrystalline 6-difluoromethyl 17oz hydroxy-4,6-pregnadiene- 3,20-dione17-acetate.

PREPARATION 3 A. 6a-IODOMETHYL-3B,17u-DIHYDROXY-5a-PREGNAN- S-ONE3,17-DIACETA'IE A suspension of 15 g. of6a-hydroxymethyl-3B,17tx-dihydroxy-Sa-pregnan-ZO-one 3,17-diacetate (seePreparation 1A) and 20 g. of triphenylphosphite methiodide in 30 ml. ofmethyl iodide was heated at refluxing temperature for a period of about2 hours or about one hour longer than the formation of a clear solution.Most of the solvent was evaporated leaving a dark syrup. The syrup wasdiluted with a mixture of methylene chloride and ether and washedsuccessively with water, dilute sodium thiosulfate, additional water anddried. The .solvent was removed with a rotary evaporator to give alight-colored oil. Addition of methanol to the oil precipitated theproduct which was collected on a filter, washed with a small amount ofmethanol and dried to yield 12.3 g. of material melting at 202 to 204 C.An analytical sample was obtained by recrystallization of the productfrom a mixture of acetone and Skellysolve B (hexanes) to yield 60ciodomethyl-3B,17a-dihydroxy-5a-pregnan-3-one 3,17- diacetate with amelting point of 201 to 205 C. and a rotation [0c] of +28 (chloroform).

Analysis.CalCd. for C26H3905I: (3, H, I, 22.72. Found: C, 56.37; H,7.04; I, 20.64.

B. 6ct-FLUOROMETHYL-3B,17a-DIHYDROXY-5a- PREGNAN-ZO-ONE 3,17-DIACETATE Asolution of silver fluoride was prepared in a polyethylene bottle bycondensing 26 g. of hydrogen fluoride, adding 200 ml. of acetonitrileand then adding an excess of silver oxide, filtering off the unreactedsilver oxide and recovering the clear silver fluoride solution. ml. ofthe thus prepared silver fluoride solution mixed with 12 g. of6ot-iodomethyl-3fi, 17oc-dihydroxy-5a-pregnan-3-0ne 3,17-diacetate wasstirred at room temperature for a period of about one-half hour and thenheated at refluxing temperature for a period of about 2 hours. Most ofthe solvent was evaporated off with a stream of nitrogen. The remainingconcentrated material was diluted with Water and extracted thoroughlywith methylene chloride. The extracts were pooled, washed with water,dried and concentrated to dryness to give a crude partly crystallineproduct containing 6a-fluoromethyl-3B,17a-dihydroxy-5apregnan-ZO-one3,17-diacetate which can, if desired, be separated by conventionalmeans, e.g., by chromatography and crystallization.

C. 6a-FLUOROMETHYL-3JS,17a.DIHYDROXY-5a- PREGNAN-ZO-ONE 17-ACETATE About9 g. of the crude partly crystalline product containing6a-fluoromethyl-3fi, 17a-dihydroxy-5ot-pregnan-20 one 3,17-diacetate(obtained in Preparation 3B) was dissolved in 200 ml. of methanol and 2ml. of concentrated hydrochloric acid and refluxed for a period of aboutan hour under a stream of nitrogen. Part of the methanol was evaporatedwith nitrogen, and water added to cause precipitation. The precipitatewas separated by filtration, washed with water and dried to yield 8 g.of a crude product containing 6a-fiuoromethyl-38,17u-dihydroxy-5otpregnan-ZO-one 17-acetate which can, if desired, bepurified by conventional means, e.g., by chromatography andcrystallization.

D. Ga-FLUOROMETHYL-l7a-HYDROXY5a-PREGNANE- 3,20-DIONE 17-ACETATE 8 g. ofthe crude product containing 6u-fiuoromethyl-3B,17a-dihydroxy-5a-pregnan 20 one 17 acetate (obtained in PreparationBC) was added to a solution of 8 g. of sodium dichromate dihydrate in 70ml. of acetic acid. After a period of about /2 hours, the reactionmixture was poured into water and the resulting precipitate filtered,washed with water and sucked nearly dry. The precipitate was dissolvedin methylene chloride and the organic phase separated and dried overmagnesium sulfate. The methylene chloride solution was adsorbed on acolumn of 300 g. of Florisil (synthetic magnesium silicate) and thecolumn extracted by gradient elution chromatography employing 4 l. of 4%acetone in Skellysolve B and 4 l. of acetone in Skellysolve B.

6a-fiuoromethyl-17u-hydroxy-5a-pregnane 3,20-dione 17-acetate was elutedin fractions 23 to 29. These fractions were pooled and evaporated; thecombined residues on recrystallization gave 2 g. of product with amelting point of 172 to 175 C. An analytical sample was obtained byrecrystallization from a mixture of acetone and Skellysolve B, providingpure 6ot-fiuoromethyl-17a-hydroxy-5a-pregnane-3,20-dione 17-acetate witha melting point of 175 to 176 C.

Analysis.-Calcd. for C H FO C, 70.90; H, 8.68; F, 4.67. Found: C, 71.08;H, 8.88; F, 4.59.

E. 6a-FLUOROMETHYL-17a-HYDROXY-4-PREGNENE- 3,20-DIONE IT-ACETATE 410 mg.of 6a-fluoromethyl-17a-hydroxy-5a-pregnane- 3,20-dione 17-acetate in 10ml. of dioxane was acidified with a drop of 4 N hydrogen bromide indioxane and 320 mg. of bromide added over a period of 1 minute. After aperiod of about 1 hour at room temperature, an excess of sodiumbicarbonate solution was added to the reaction mixture. The precipitated2,4-dibromo derivative of6afluoromethyl-17u-hydroxy-5a-pregnan-3,20-dione 17-acetate was treatedwith 0.9 g. of sodium iodide in ml. of acetone containing bromoacetone,and the mixture heated at refluxing temperature for a period of about2.5 hours. 0.3 g. of oxalic acid was then added and heating continuedfor a period of about 1 hour. After cooling, ethyl acetate was added andthe solution filtered. The filtrate was washed with water and sodiumbicarbonate solution, then dried with sodium sulfate. The filtrate wasstirred with 500 mg. of zinc dust in 2 ml. of acetic acid for about 1hour and then filtered. The organic layer was washed successively withwater, sodium bicarbonate solution and dried with sodium sulfate.Evaporation of the solvent gave the crude a,/8-unsaturated ketone, whichon purification with a Girard reagent, followed by subsequentcrystallization yielded pure light colored6u-fluoromethyl-17uhydroxy-4-pregnene-3,20-dione 17-acetate.Alternatively, if desired, the crude a,B-unsaturated ketone can bepurified by chromatography over Florisil with increasing proportions ofacetone in Skelysolve B, followed by recrystallization.

F. 6a-FLUOROMETHYL-l7a-HYDROXY-4-PREGNENE- 3,20-DIONE 17-ACETATE Amedium consisting of 1% dextrose hydrate, 2% cornsteep liquor of 60%solids and tap Water was adjusted to pH 4.9 with sodium hydroxide. Themedium was steam sterilized at 15 pound pressure for about 30 minutes,cooled and then inoculated with a 24 hour growth, from spores, ofNocardia blackwellii (NCTC 630 [Medical Research Council of the ListerInstitute, London]). The medium was agitated and sparged with sterileair at the rate of one-tenth volume of air per volume of medium perminute. At the end of 24 hours of fermentation at room temperature, thepH was about 7.4. To this culture, there was added a solution of60t-fll10l'0- methyl-l7oc-hydroxy-5a-pregnane 3,20 dione 17-acetatedissolved in a minimal amount of dimethylformamide.

The solution was prepared by dissolving 5 parts of the steroid in 100parts of the solvent and adding about 10 ml. of the solution per literof the medium. Fermentation was continued for a period of about 6 hourswhereupon the mycelium and beer Were extracted thoroughly with methylenechloride. The extract was washed with sodium bicarbonate solution andthen with water, dried and concentrated under vacuum to give6u-fiuoromethyl-17a-hydroxy-4-pregnene-3,20-dione.

Instead of Nocardia blackwellii (NCTC 630 [Medical Research Council ofthe Lister Institute, London]) used to produce fermentativedehydrogenation at the 4,5-position, other microorganisms may besimilarly elfectively employed; included are those chosen from the groupconsisting of: ATCC 4275 (Nocardia convoluta); ATCC 9604 (Nocardiagardneri) and NRRL 13-1365 (Nocardia coeliaca).

A solution composed of 1 g. of6u-fluoromethyl-17uhydroxy-4-pregnene-3,20-dione 17-acetate, 2.5 ml. ofdistilled acetic anhydride, 250 mg. of p-toluenesulfonic acid and 2.5m1. of acetic acid was stirred for a period of about minutes. Themixture was poured with vigorous stirring into water. The precipitatedsolid was separated by filtration, dried, chromatographed over Florisilwith increasing proportions of acetone in Skellysolve B andrecrystallized from ethyl acetate to yield light-colored 6afiuoromethyl17cc hydroxy-4-pregnene-3,20-dione 17- acetate.

PREPARATION 4 6-fluoromethyl-1 7-lzydr0xy-4,6-pregnacliel1e-3,20- dione1 7-acetate 1 g. of 6a-fluoromethyl-l7u-hydroxy-4-pregnene-3,20- dione17-acetate, 1.5 g. of recrystallized2,3,5,6-tetrachl-oro-l,4-benzoquinone (chloranil) and 60 ml. of tertiaryamyl alcohol were heated to boiling under nitrogen with a few boilingchips, and gently refluxed for a period of about 6 hours. The mixturewas cooled and evaporated to dryness under reduced pressure. The solidresidue (with the exception of some chloranil, which was insoluble) wasdissolved in about ml. of ether and filtered. The chloranil on thefilter paper was washed with several portions of ether and the combinedether filtrates washed with 200 ml. portions of cold 2% sodiumhydroxide. The ether filtrates were washed with cold water until thewashings were neutral, then with saturated sodium chloride solution. Thepooled ether solutions were dried over sodium sulfate and evaporated todryness. The residue crystallized readily from cold acetone to yieldpure light-colored crystalline6-fluoromethyl-17ahydroxy-4,6-pregnadiene-3,20-dione 17-acetate.

PREPARATION 5 3,6,1 7a-dilzydroxy-4-pregnen-ZO-one 1 7-acetate To asolution of 10 g. of 17a-hydroxy-4-pregncne-3,20- dione 17-acetate in250 ml. of purified tetrahydrofuran, cooled to between 5 C. to 15 C.,there is added in small portions and with stirring 20 g. of lithiumaluminum tri-t-butoxyhydride. The reaction mixture is allowed togradually come to room temperature and the excess lithium aluminumtri-t-butoxyhydride is destroyed by the addition of dilute acid. Thereaction mixture is washed with dilute hydrochloric acid, dried andchromatographed over a Florisil (synthetic magnesium silicate) columnpacked wet with commercial hexanes. The column is eluted with commercialhexanes containing increasing amounts of acetone and those fractionswhich by thin layer chromatography and ultraviolet absorption show thepresence of the desired product are taken to dryness and recrystallizedfrom mixtures of acetone-water and acetone-commercial hexanes to yield35,17a-dihydroxy- 4-pregnen-20-one l7-acetate, a crystalline solid.

3 3,l7a-dihydroxy-6u-fluoro-4-pregnen-2O-one S-hemisuccinate l7-acetate,3/3,l7a-dihydroxy-Ga-chloro-4-pregnen-20-one 3-hemisuccinate 17-acetate,35,17u-dihydroxy-6a-trifluoromethyl-4-pregnen-20-one 3-hemisuccinatel7-acetate, 35a,17a-dihydroxy-6a-difluoromethyl-4-pregnen-ZO-one3-hernisuccinate l7-acetate, 3f,17a-dihydroxy-6a-fluoromethyl-4-pregnen-20-one 3-hemisuccinate17-acetate, 3 ,B, 17 a-dihydroxy- 1 6-methylene-4-pregnen-ZO-one3-hemisuccinate 17-acetate, 3 5,17 ot-dihydroxy-6a-methyl- 16-methylene-4-pregnen- 20-one 3-hemisuccinate 17-acetate,3,8,17a-dihydroXy-6a,16a-dimethyl-4-pregnen-2O-one 3-hemisuccinate17-acetate, 3 3, 17 a-dihydroxy-6u, l 7 fi-dimethyl-4-pregnen-20-oneS-hemisuccinate 17-acetate, 3B,17ot-dihydroxy-4,6-pregnadiene-20-one3-hemisuccinate 17-acetate,3,3,17a-dihydroxy-6-methlyl-4,6-pregnadien-20-one 3-hemisuccinate17-acetate, 3(3,17a-dihydroxy-6-fluoro-4,6-pregnadien-ZO-one3-hemisuccinate 17-acetate,35,l7a-dihydroxy-6-chloro-4,6-pregnadien-20-one S-hernisuccinate17-acetate, 3,6,17ot-dihydroxy-6-trifluoromethyl-4,6-pregnadien- 20-oneB-hemisuccinate l7-acetate,3p,17a-dihydroxy-6-difluoromethyl-4,6-pregnadien- 20-one S-hemisuccinate17-acetate, 3 3,l7a-dihydroxy-6-fiuoromethyl-4,G-pregnadien- 20-one,3-hemisuccinate 17-acetate,3B,17a-dihydroxy-16-methylene-4,6-pregnadien-20- one 3-hemisuccinate17-acetate,3,6,17a-dihydroxy-6-methyl-16-methylene-4,6-pregnadien-ZO-one,B-hemisuccinate 17-acetate,3p,17a-dihydroxy-6,16a-dimethyl-4,6-pregnadien- 20-one 3-hemisuccinate17-acetate, and 3,8,17u-dihydroxy-6,16B-dimethyl-4,6-pregnadien-20- one3-hemisuccinate 17-acetate, respectively.

Similarly, reacting the 35,17e-dihydroxy steroid 17-acetate startingmaterials with the appropriate hydrocarbon dicarboxylic acid, or theacid anhydrides or acid halides thereof, is productive of thecorresponding 3-acylates, such as, for example, the 3-hemimalonate, theB-hemiglutarate, the 3-hemiadipate, the 3-hemipimelate, the 3-hemisuberate, the 3-hemi-a,B-dimethylsuccinate, the 3-hemi-a,fl-diethylsuccinate, the 3-hemi-fi-methylglutarate, the3-herni-a,;8-dimethylglutarate, the 3-hemi-a,a-diethylglutarate, the3-hemi;3,,B-dirnethylglutarate, the 3-hemi-aethyl-B-methylglutarate, the3hemi-18-ethyl-fl-methylglutarate, the 3-hemi-a,a,}8-trimethylglutarate,the S-hemia,B,fl-trimethylglutarate, the 3-hemi e 9, trimethylglutarate,the 3-hemi-a-ethyl-B,fl-dimethylglutarate, the 3-hemi-u,B,fi-tetramethylglutarate, the3-hemi-ot,;3,}3,'y-tetramethylglutarate, the3-hemi-fi-methyl-B-butylglutarate, the3-hemi-j3-methyl-fi-isobutylglutarate, the B-hemi-a-methyladipate, the3-hemi-a,ot-dimethyladipate, the 3-hemimaleate, the 3-hemi-citraconate,the 3-hemi-(o-, m-, pphthalate), the 3-hemi-diglycolate, and the like,of 35,17a-dihydroxy-4-pregnen-20-one 17-acetate,3e,17a-dihydroxy-6a-methyl-4-pregnen-20-one 17-acetate,318,17a-dihydroxy-6a-fiuoro-4-pregnen-ZO-one 17acetate,3,8,l7ot-dihydroxy-6a-chloro-4-pregnen-20-one 17-acetate,3,8,17a-dihydroxy-6a-trifiuoromethyl-4-pregnen-ZO-one l7-acetate,

3 ,8, 17 a-dihydroxy-6ot-difluoromethyl-4-pregnen-20-one 17-acetate,

3p,17a-dihydroxy-6e-fluoromethyl-4-pregnen-ZO-one l7-acetate,

3 ,8, 17 u-dihydroxyl6-methylene-4-pregnen-20-one 17-acetate,

3,8,l7u-dihydroxy-6a-methyl-16-methylene-4-pregnen- 20-one 17-acetate,

1 6 3fl,l7oc-dihydroxy-6a,l6a-dimethyl-4-pregnen-20-one l7-acetate,3p,17a-dil1ydr0xy-6a,16B-dimethyl-4-pregnen-20-one 17-acetate,313,17u-dihydroxy-4,6-pregnadien-20-one l7-acetate,3B,17oc-dihydroxy-6-methyl-4,6-pregnadien-ZO-one l7-acetate,318,17a-dihydroxy-6-fluoro-4,6-pregnadien-20-one.

l7-acetate, 35,17a-dihydroxy-6-chloro-4,6-pregnadien-20-one 17-acetate,1 3,8,17a-dihydroxy-6-triflu0romethyl-4,6-pregnadien-20- one 17-acetate,3B,l7or-dihydroxy-6-difiuorornethyl-4,6-pregnadien-20- one 17-acetate,3B,17a-dihydroxy-6-fluoromethyl-4,6-pregnadien-20-one 17-acetate,3,8,l7a-dihydroxy-16-methylene-4,6-pregnadien-ZO-one 17-acetate,3e,17a-dihydroxy-6-methyl-16-methylene-4,6-pregnadien- 20-one17-acetate, 313,17a-dihydroxy-6,16a-dimethyl-4,6-pregnadien-20-one17-acetate, and 3B,17a-dihydroxy-6,16,8-dimethyl-4,6-pregnadien-20-one17-acetate.

In the above procedures the esterifying agent is preferably theanhydride. However, when the esterifying agent is the free acid, thereaction is carried out in the presence of an esterification catalyst.

If the corresponding acylating agent is solid, an inert solvent such astoluene, methylene chloride, dimethylformamide or dioxane can be addedif desired to effect solution and to provide a liquid esterificationmedium.

Where the esterification reaction does not proceed to completion at roomtemperature, the temperature can be raised from room temperature to ashigh as 90 C. The course of the reaction is conveniently followed bythin layer chromatography on alumina or Florisil (synthetic magnesiumsilicate) using disappearance of the 3-hydroxy starting material as theendpoint. Hindered anhydrides of hydrocarbon carboxylic acids react moreslowly than succinic anhydride and, if incomplete reaction at roomtemperature is observed after 24 hours, the temperature is raised,preferably to about C., and the reaction permitted to proceed tocompletion.

EXAMPLE 2 35,17a-dihydroxy-4-prregnen-20-0ne 3-s0dium succinate17-acetate (III) 1.0 g. of 3p,17a-dihydroxy-4-pregnen-20-one3-hemisuccinate 17-acetate is dissolved in 25 ml. of acetone andfiltered through a medium porosity glass filter. The thus obtainedsolution is cooled in an ice bath and there is added thereto a filteredaqueous solution of exactly one equivalent (0.177 g.) of sodiumbicarbonate. The resulting solution is concentrated under house vacuum,at a temperature not exceeding 25 C., to remove the acetone andlyophilized under high vacuum to yield 35,17a-dihydroxy-4-pregnen-20-one3-sodium succinate 17-acetate, a fluffy white solid which is very watersoluble.

In like manner, substituting a stoichiometric equivalent amount of3,8,17u-dihydroxy-6e-methyl-4-pregnen-20-one 3-hemisuccinate 17-acetate,

3B,17u-dihydroxy-6a-fiuoro-4-pregnen-20-one 3-hemisuccinate 17-acetate,

3e,17a-dihydroxy-6a-chloro-4-pregnen-20-one 3-hemisuccinate 17-acetate,

3p,17a-dihydroxy-6ot-trifiuoromethyl-4-pregnen-20-one S-hemisuccinate17-acetate,

3 8,17a-dihydroxy-6a-difluoromethyl-4-pregnen-20-one 3-hemisuccinate17-acetate,

3p,17a-dihydroxy-6a-fluoromethyl-4-pregnen-20-one 3-hernisuccinate17-acetate,

23 3fl,17a-dihydroxy-6-methyl-16-methylene-4,6-pregnadien- 20-one3-diethylarninoacetate 17-acetate, 3B,17a-dihydroxy-6,l6u-din1ethyl-4,6-pregnadien-ZO-one 3-diethylaminoacetate l7-acetate, and3/3,17u-dihydroxy-6,16[3-dimethyl-4,6-pregnadien-ZO-one3-diethylaminoacetate 17-acetate,

for 3,8,17a-dihydroxy-4-pregnen-20-one 3-diethylarninoacetate l7-acetateis productive of Sfi,17oc-dihydroxy-6a-methyl-4-pregnen-ZO-one3-diethylaminoacetate 17-acetate hydrochloride,

3,8,17u-dihydroxy-6a-fluoro-4-pregnen-20-one 3-diethylaminoacetate17-acetate hydrochloride,

3[i,17a-dihydroxy-6ot-chloro-4-pregnen-20-one 3-diethy1- aminoacetatel7-acetate hydrochloride,

35,17a-dihydroxy-6a-trifiuoromethyl-4-pregnen-20-one3-diethylaminoacctate 17-acetate hydrochloride,

35,17 a-dihydroxy-6a-difluoromethyl-4-pregnen-20-0ne 3-diethylaminoacetate l7-acetate hydrochloride,

35,17a-dihydroxy-6a-fluoromethyl-4-pregnen-ZO-one 3-diethylaminoacetatel7-acetate hydrochloride,

313,l7a-dihydroxy-16-methylene-4-pregnen-20-one 3-diethylaminoacetate17-acetate hydrochloride,

3B,17oc-dihydroxy-6a-methyl-16-methylene-4-pregnen-20- one3-diethylaminoacetate 17-acetate hydrochloride,

3,8,17a-dihydroxy-6a,16a-dimethyl-4-pregnen-ZO-one 3-diethylaminoacetate 17-acetate hydrochloride,

3,8,17a-dihydroxy-6a,16/3-di=methyl-4-pregnen-20-one3-diethylaminoacetate l7-acetate hydrochloride,

3,8,17a-dihydroxy-4,6-pregnadien-20-one 3-diethylaminoacetate 17-acetatehydrochloride,

35,17a-dihydroxy-6-rnethyl-4,6-pre-gnadien-20-one 3-diethylaminoacetate17-acetate hydrochloride,

3p,l7a-dihydroxy-6-fluoro-4,'6-pregnadien-20-0ne 3-diethylaminoacetate17-acetate hydrochloride,

3B,17a-dihydroxy-6-chloro-4,6-pregnadien-20-one 3-diethylaminoacetate17-acetate hydrochloride,

3B,l7urdihydroxy-6-trifluoromethyl-4,6-pregnadien- 20-oneB-diethylaminoacetate 17-acetate hydrochloride,

3/8,17a-dihydroxy-6-difluoromethy1-4,6-pregnadien- 20-one3-diethylaminoacetate l7-acetate hydrochloride,3p,l7u-dihydroxy-6-fluoromethyl-4,6-pregnadien-20- one3-diethy1aminoacetate 17-acetate hydrochloride,3,8,17a-dihydroxy-16-methylene-4,6-pregnadien-20-oneB-diethylaminoacetate 17-acetate hydrochloride,3,8,l7m-dihydroxy-6-methyl-16-methylene-4,6-pregnadien-20-0ne3-diethylaminoacetate 17-acetate hydrochloride,

3 ,8, l7vt-dihydroxy-6, l 6a-dimethyl-4,6-pregnadien-20- one3-diethylaminoacetate 17-acetate hydrochloride, and

313,17u-dihydroxy-6,16fl-dimethyl-4,6-pregnadien-20- one3-diethylaminoacetate l7-acetate hydrochloride, respectively.

Similarly, reacting the 3,8,l7a-dihydroxy steroid 3- aminoacetate17-acetate starting materials with hydrochloric acid is productive ofthe corresponding hydrochloride salt of the 3-arninoacetates, such as,for example, the 3-dimethylamin0acetate, the 3-dipropylarninoacetate,the 3-dioctylaminoacetate, the 3-(methylethylaminoacetate), the3-(methylpropylaminoacetate), the 3-dibenzylaminoacetate, the3-(methylbenzylaminoacetate), the 3- pyrrolidinoacetate, the3-piperidinoacetate, the 3-morpholinoacetate, and the like, of

3,8,17m-dihydroxy-4-pregnen-20-one l7-acetate,

3/3,l7a.-dihydroxy-6oumethyl-4-pregnen-20-one l7- acetate,

36,17a-dihydroxy-6a-fluoro-4-pregnen-20-one 17- acetate,

3/3,17a-dihydroxy-6a-chloro-4-pregnen-20-one 17- acetate,

24 3p,17a-dihydroxy-6a-tri-fluoromethyl-4-pregnen-20 -one 17-acetate, 3,8, l7a-dihydroxy- 6u-difluoromethyl-4-pregnen-20-one 17-acetate,3p,17a-dihydroxy-6arfluoromethyl-4-pregnen-20-one l7-acetate,3,8,17a-dihydr0xy-16-methylene-4-pregnen-ZO-one 17- acetate, 3 l3,l7a-dihydroxy-6a-methyl-l 6-methylene-4-pregnen- 20-one l7-acetate,313,l7a-dihydroxy-6a,16u-dimethyl-4-pregnen-20-one 17-acetate,3;3,17u-dihydroxy-6u,l6fl-dimethyl-4-pregnen-20-one 17-acetate,3,8,17m-dihydroxy-4,6-pregnadien-20-one l7-acetate,3p,17u-dihydroxy-6-methyl-4,6-pregnadier1-20-one 17- acetate,35,17a-dihydroxy-6-fluoro-4,6-pregnadien-20-one 17- acetate,3p,17a-dihydroxy-6-chloro-4,6-pre-gnadien-20-one 17- acetate, 3 B, 17a-dihydroxy-6-trifluoromethyl-4,6-pregnadien- 20-0ne 17-acetate,3p,17a-dihydroxy-6-difluoromethyl-4,6-pregnadien-20- one l7-acetate,35,l7ot-dihydroxy-6-fluoromethyl-4,6-pregnadien-20- one 17-acetate,3B,17a-dihydroxy-16-rnethylene-4,6-pregnadien-20-one 17-acetate, 3 p, 17a-dihydroxy-6-Inethyl-1 6-methylene-4,6-pregnadien-20-one 17-acetate,3/3,17ardihydroxy-6,16a-dimethyl-4,6-pregnadien-20- one 17-acetate, and3/3,17a-dihydroxy-6,16;8-dimethy1-4,6-pregnadien-ZO- one 17-acetate.

Likewise, substituting a stoichiometric equivalent amount of theappropriate pharmacologically acceptable acid, such as, for example,formic, acetic, propionic, hydrobromic, hydriodic, sulfonic, phosphoric,nitric, lactic, pamoic, and the like, for hydrochloric acid, isproductive of the corresponding formate, acetate, propionate,hydrobrornide, hydriodide, sulfate, phosphate, nitrate, lactate,parnoate, and the like, of the 3-aminoacetate steroids of this example,Example 5.

EXAMPLE 6 3,8,1 7a-dihydroxy-4-pregnen-20-one 3-diethylaminoacetate17-acetate methobromide (VII) 500 mg. of3,8,l7a-dihydroxy-4-pregnen-20-one S-diethylaminoacetate 17-acetate isdissolved in 5 ml. of cold benzene. The solution is chilled and to it isadded 0.5 ml. of methylbromide. The flask is sealed and maintained atroom temperature for 72 hours. The resulting reaction mixture isconcentrated to a small volume and diluted with ether. There is thusprecipitated 313,17a-dihydroxy- 4 pregnen 2O one 3 diethylaminoacetate17 acetate methobromide.

In like manner, substituting a stoichiometric equivalent amount of35,17u-dihydroxy-6wmethyl-4-pregnen-20-one 3-diethylaminoacetate17-acetate, 3p,l7a-dihydroxy-6ot fluoro-4-pregnen-20-one3-diethylaminoacetate 17-acetate,3/3,17u-dihydroxy-6archloro-4-pregnen-ZO-one 3-diethylaminoacetate17-acetate, 3B,17a-dihydroxy-6a-trifluoromethyl-4 pregnen-20- one3-diethylaminoacetate l7-acetate, 35,17a-dihydroxy-6x-difluoromethyl-4-pregnen-20- one 3-diethylaminoacetate 17-acetate,3p,17a-dihydroxy-6a-fluoromethyl-4-pregnen-20- one 3-diethylaminoacetate17-acetate, 3 B,l7a-dihydroxy-16-methylene-4-pregnen-20-one3-diethylaminoacetate 17-acetate,

313,17a-dihydroxy-6-difluoromethyl-4,6-pregnadien-20-one 3-chloroacetatel7-aceta-te, 3p,17a-dihydroxy-6-fluoromethyl-4,6-pregnadien-20-one3-chloroacetate 17-acetate,3p,Hot-dihydroxy-16-methylene-4,6-pregnadien-ZO-one 3-chloroacetate17-acetate,

3,8,17a-dihydroxy-6-methyl-16-methylene-4,6-pregnadien- 20-one3-chloroacetate l7-acet-ate, 3,8,l7a-dihydroxy-6,16a-dimethyl-4,6-pregnadien-20-one 3-chloroacetate17-acetate, and 3,8,l7a-dihydroxy-6,l6,8-dimethyl-4,6-pregnadien-20-one3-chloroacetate 17-acetate,

for 313,17u-dihydroxy-4apregnen-20-one 3-chloroacetate -17-acetate isproductive of 3p,17a-di'hydroxy-6a-methyl-4-pregnen-ZO-one3-pyridiniumacetate l7-acetate chloride,3p,17u-dihydroxy-6a-fiuoro-4-pregnen-20-one 3-pyr-idiniumacetate17-acetate chloride, 33,17a-dihydroxy-6a-chloro-4-pregnen-20-one3-pyr-idiniumacetate 17-acetate chloride, 3 8,17a-dihydroxy-6a-t-rifluoromethyl-4-pregnen-20-one 3-pyridiniumacetatel7-acetate chloride, 3p,17a-dihydroxy-6a-difluoromethyl-4-pregnen-20-one3-pyridinium-acetate 17-acetate chloride, 3 ,3, l7ot-dihydlOXY-6oc-flll0l01'116EhY1-4-PIC gnen-ZO-one 3-pyridiniumacetate17-acetate chloride, 3,9,17u-dihydroxy-16-methylene-4-pregnen-20-one 3pyridiniumacetate 17-acetate chloride,3p,l7a-dihydroxy-6a-methyl-l6-methylene-4-pregnen-20- one3-pyridiniumacetate 17-acetate chloride,3fl,17a-dihydroxy-6a,16a-dimethyl-4-pregnen-20-one B-pyridiniumacetate17-acetate chloride, 35,17a-dihydroxy-6a,l6B-dimethyl-4-pregnen-20-oneB- yridiniumacetate 17-acetate chloride, 38,17a-dihydroxy-4,6-pregnadien-ZO-one 3-pyridinumacetate l7-acetatechloride, 3p,17a-dihydroxy-6-methyl-4,6-pregnadien-20-one3-pyridiniumacetate 17-acetate chloride, 318,l7a-dihydroxy6-tfiuoro-4,6pregnadien-20-one 3-pyridiniumacetate 17-acetate chloride, 33,17u-dihydroxy-6-chloro-4,6-pregnadien-20-one 3-pyridiniumacetate17-acetate chloride,3,6,17a-dihydroxy-6-trifluoromethyl-4,6-pregnadien-20- one B-yridiniumacetate 17-acet-ate chloride,3p,17tat-dihydroxy-6-difluoromethyl-4,6-pregnadien-20- one3-pyridiniumacetate 17-acetate chloride,313,l7a-dihydroxy-6-fiuoromethyl-4,6-pregnadien-ZO-one3-pyridiniumacetate 17-acetate chloride,

23 3 3,l7a-dihydroxy-16-methylene-4,6-pregnadien-20-one3-pyridiniumacetate l7-acetate chloride,3,8,17u-dihydroxy-6-methyll6-methy1ene-4,6-pregnadien- 20-one3-pyridiniumacetate 17-acetate chloride,3fl,l7a-dihydroxy-6,16a-dimethyl-4,6-pregnadien-20-one3-pyridiniumacetate 17-acetate chloride, and35,17u-dihydroxy-6,l6p-dimethyl-4,6Hpregnadien-2O-One3-pyridiniumacetate 17-acetate chloride, respectively.

Similarly, substituting a stoichiometric equivalent amount of the3-bromoacetate of the 3;8,l7a-dihydroxy steroid 17-acetates for theS-chloroacetates given above is productive of the corresponding 3fi,l7a-dihydroxy steroid 3-pyrid'iniumacetate 17-acetate bromides.

The 3-pyridinium bromides and chlorides of this example have the sameuses and are administered in the same manner as the quaternary ammoniumsalts of the 3- aminoacetates described above.

EXAMPLE 8 313,1 7 a-dihydroxy-6 a-methyl-4-pregnen-20-one3-hemisuccinate 17-acetate (II) To a solution of 2.5 g. of3p,17a-dihydroxy-6u-methyl- 4-pregnen-20-one 17-acetate in 25 ml. ofpyridine there was added 2.5 g. of succinic anhydride. The reactionmixture was kept at room temperature for about 16 hours and then heatedat 60-70 C. for about 12 hours. The succina-te ester appeared as theonly spot near the origin when a small amount of the reaction mixtureWas subjected to thin layer chromatography. The reaction mixture waspoured into ice water containing 30 ml. of concentratcd hydrochloricacid. The precipitate which formed was collected, washed well withwater, dried, slurried with ml. of water and a small amount ofdeter-gent (to held wet the precipitate), followed by the slow additionof 60 ml. of 0.1 N sodium hydroxide to effect solution of theprecipitate. Filtration through a bed of Celite (diatomaceous earth)removed the small amount of insoluble matter. The filtrate obtained wasreprecipitated by the dropwise addition of 3 N hydrochloric acid. Theprecipitate which formed during the recrystallization was collected,washed with water, and dried to yield 2.7 g. of product comprising35,17a-dihydroxy-6a-methyl-4-pregnen-20-one 3-hemisuccinate 17- acetate,which was used in the following example (EX- \mple 9) without furtherpurification.

EXAMPLE 9 313,1 7 oc-dihydr0xy-6 a-methyl-4-pregnen-2 0-0ne 3 -s0di umsuccinate 1 7 aace'tate (II I Water was intermittently added while theacetone wasremoved on a rotary evaporator. Following removal of theacetone a clear solution was obtained which was diluted to about 15 ml.with water and swirled while freezing in a one liter round bottom flask.The frozen product was dried on a lyophilizer and the fluffy amorphouspowder obtained was dissolved in water and filtered through a bed ofCelite (diatomaceous earth) to remove the scum which had formed. Thefiltrate was freeze-dried again to give 1.0 g. of 3B,17u-dihydroxy- 60cmethyl 4 pregnen 20 one 3-sodium succinate 17-acetate, an amorphoussolid, having intense infrared absorptions at 1725 and 1245 cmr Themolecular weight as determined by titration was 529 (510 theory).

EXAMPLE 10 3 ,8,] 7 a-dihydrxy-6 a-m ethyl-4 pregnen-2 O-one3-chl0r0acetate 17-acetate (IV) A slow stream of nitrogen was passedthrough a solution of 2.0 g. of 35,170; dihydroxy 60c methyl 4-pregnen-ZO-one 17-acetate in 5 ml. of pyridine. The solution was cooledin an ice-salt bath and a solution of 1.0 g. of chloroacetic anhydridein 30 ml. of dry ether was added dropwise with stirring. After 3 hoursmost of the ether had been expelled by the nitrogen stream. Thin layerchromatography of a small portion of the reaction mixture showed theabsence of starting material. Ten drops of water was added to destroythe excess chloroacetic anhydride. The solution was poured intoice-water and extracted with ether. The ether extracts were combined,washed with cold dilute hydrochloric acid, water, dilute cold sodiumbicarbonate, and again with water, dried over sodium sulfate, followedby removal of the ether by evaporation. The residue remaining wasrecrystallized from ether-commercial hexanes to give 1.9 g. of 3fi,l7adihydroxy 6a methyl- 4 pregnen 20 one 3 chloroacetate 17 acetate havinga melting point of 123 126 C., [a] 19 (CHCl and the following analysis:

Analysis.Calcd. for C H C1O C, 67.15; H, 8.02; Cl, 7.62. Found: C,67.28; H, 8.18; Cl, 7.70.

EXAMPLE 11 35,17a-dihydr0xy-6a-methyl-4-pregnen-20-oneS-diethylaminoacetate 17-acetaze (V) A solution of 0.75 g. of 3fi,17ocdihydroxy-6oc-methyl- 4-pregnen-20-one 3-chloroacetate 17-acetate in ml.of tetrahydrofuran and 2 ml. of diethylamine was heated at 60-65 C. in asealed tube for two hours and then at 50 C. for 18 hours. The solutionwas cooled and the crystals of diethylamine hydrochloride were removedby filtration. The filtrate was then concentrated to dryness (to removetraces of diethylamine) and the residue was dissolved in ether. Theether was removed by evapora tion to yield an amorphous residue of3fl,17a-dihydroxy- 6a methyl 4 pregnen 20 one 3 diethylaminoacetate17-acetate which was dried under vacuum and used in the followingexample (Example 12) without further purification.

EXAMPLE 12 3 5,1 7 a-dihydroxy-o a-methyl-4-pregnen-20-one3-diethylaminOacetate 1 7 -acetate hydrochloride (VI) The amorphousresidue of 3,8,17a dihydroxy 60cmethyl 4 pregnen 20 one 3diethylaminoacetate 17-acetate, obtained in Example 11, was dissolved in30 ml. of absolute ether and 4.5 ml. of a freshly prepared 0.39 Netheral hydrogen chloride solution was added dropwise. The precipitatedamorphous solid was filtered, washed with ether and dried to yield 0.8g. of 35,170:- dihydroxy 6a methyl 4 pregnen-ZO-one3-diethylaminoacetate 17-acetate hydrochloride having infraredabsorption bands at 2700, 2510, 2440, 1745, 1735, 1700, 1660, 1245 and1210 cm." and the following analysis:

Analysis.Calcd. for C H O NCl: C, 66.95; H, 8.99; N, 2.60; CI, 6.59.Found: C, 66.93; H, 9.18; N, 2.59; CI, 6.74.

EXAMPLE 13 Intravenous preparation A batch of 3,8,l7a dihydroxy 60cmethyl-4-pregnen- 2.0-one 3-sodium succinate 17-acetate powder issterilized with ethylene oxide gas and then 100 mg. quantities aretransferred aseptically into sterile five cubic centimeter ampuls whichare then sealed.

For emergency use in threatened abortion, sterile sodium chlorideinjection solution is added to dissolve the 100 mg. of powder and enoughvehicle used to make 30 2 ml. of solution. This solution is thenadministered intravenously or intramuscularly.

Similarly, the other steroids produced as products in Examples 2, 5, 6,7 and 12 can be substituted for the active ingredient of the formulationof Example 13.

EXAMPLE 14 Table'ls Ten thousand scored tablets for oral use, eachcontaining 5 mg. of 313,17a dihydroxy-6a-methyl-l6-methylene-4-pregnen-20-one 3-diethylaminoacetate l7-acetate hydrochloride, areprepared from the following ingredients:

Gm. 35,170: dihydroxy 6a methyl 16 methylene- 4 pregnen 20 one 3diethylaminoacetate l7- acetate hydrochloride 50 Lactose 2000 Cornstarch 300 Talc 300 Calcium stearate 40 RiO CH3 on,

on on 0 on i on 3 --OAc 3 ---OAc Ton, Torr,

RrO- R10 CH3 CH3 CH3 (3113 on 1 =0 OH R 3 --OAc 3 -0Ac R R and RrO- R1O(3H, CH3

wherein Ac is the acetyl radical, X is selected from the groupconsisting of hydrogen, fluoro, chloro, trifluoromethyl, difluoromethyl,and fluoromethyl, X is selected from the group consisting of hydrogenand methyl, R is selected from the group consisting of and R is selectedfrom the group consisting of (i) The acyl radical of a hydrocarbondicarboxylic acid containing from 3 to 12 carbon atoms, inclusive, and

(ii) An aminosubstituted acetyl radical of the formula wherein R isselected from the group consisting of the pyrrolidino, piperidino andmorpholino radicals and a radical of the formula wherein R and R areselected from the group consisting of the benzyl radical and lower alkylradicals containing from 1 to 4 carbon atoms, inclusive;

(b) The alkali metal salts of (a) (i); and

(c) The pharmacologically acceptable acid addition and quaternaryammonium salts of (a) (ii).

2. 35,170; dihydroxy 6a methyl 16 methylene- 4-pregnen-20-one3-hemisuccinate 17-acetate.

3. 3B,17oz dihydroxy 6 methyl 16 methylene- 4,6-pregnadien-20-one3-hemisuccinate 17-acetate.

4. 35,170; dihydroxy 6a,16a dimethyl 4 pregnen- 20-one 3-hemisuccinate17-acetate.

5. 3B,17cc dihydroxy 6,160: dimethyl 4,6 pregnadien-20-one3-hemisuccinate 17-acetate.

6. 3,3,1704 dihydroxy 6 chloro 4,6 pregnadien- 20-one 3-hemisuccinate17-acetate.

7. 35,170: dihydroxy 60c fluoro 4 pregnen-ZO-one 3-hemisuccinate17-acetate.

8. 35,170; dihydroxy 60 chloro-4-pregnen-20-one 3-hemisuccinate17-acetate.

9. 35,170: dihydroxy 4 pregnen-ZO-one 3-hemisuccinate 17-acetate.

10. 313,17oz dihydroxy 6 methyl 16 methylene- 4,6-pregnadien-20-one 3hemi [3,;8 dimethylglutarate 17-acetate.

11. 3fl,17a dihydroxy 6 methyl-16-methylene-4,6- pregnadien-ZO-one3-diethylaminoacetate 17-acetate.

12. A compound selected from the group consisting of35,17a-dihydroXy-6a-methyl-16-methylene-4-pregnen-20- one 3-sodiurnsuccinate 17-acetate,

3,8, 17a-dihydroXy-6-methy1-1 6-methy1ene-4,6-pregnadien- 20-one3-sodium succinate 17-acetate,35,17a-dihydroxy-6a,16a-dimethyl-4-pregnen-20-one 3- sodium succinate17-acetate, 35,17a-dihydroXy-6,16a-dimethyl-4,6-pregnadien-ZO-one3-sodium succinate 17-acetate, 3 ,8,17a-dihydroxy-6-chloro-4,6-pregnadien-20-one 3- sodium succinate17-acetate, 3B,]7a-dihydroxy-6a-fluoro-4-pregnen-20-one 3-sodiumsuccinate 17-acetate, 313,17a-dihydroxy-6u-chloro-4-pregnen-20-one3-sodium succinate 17-acetate, 313,17a-dihydroxy-4-pregnen-20-one3-sodiurn succinate 17-acetate, and3p,17a-dihydroxy-6-methyl-16-methylene-4,6-pregnadien- 20-one 3-sodil lfifi-dimethylglutarate l7-acetate.

13. 3[3,17a dihydroxy 6a methyl 4 pregnen-20- one 3-diethylaminoacetatel7-acetate hydrochloride.

14. The B-hemidiglycolate of a compound selected from the groupconsisting of those having the formulae:

CH3 CH3 CH3 CH3 41:0 dl=0 CH3 --OAc ---0Ac 1 -01 Ton,

no no 7 l X1 X1 CH: CH3

CH3 CH:

:0 :0 CH3 k0 0Ac and wherein Ac is the acetyl radical, X is selectedfrom the group consisting of hydrogen, fluoro, chloro, trifl-uoromethyl,difluoromethyl, and fluoromethyl X is selected from the group consistingof hydrogen and methyl and R is selected from the group consisting of15. The 3-pyridiniumacetate halide, wherein the halide is selected fromthe group consisting of chloride and bromide, of a compound selectedfrom the group consisting of those having the formulae:

ona

and H- and 1 6. A compound selected from the those having the formulae:

group consisting of wherein Ac is the acetyl radical, X is selected fromthe group consistingof hydrogen, methyl, fluoro, chloro,trifluor-omethyl, difluoromethyl, and fiuoromethyl, X is selected fromthe .group consisting of hydrogen and methyl, R is selected from thegroup consisting of and and Z is selected from the group consisting ofchlorine and bromine.

17. 3,8,17a-dihydnoxy-6a-methyl-4-pregnen-20-one 3- chloroacetatel7-acetate.

18. Process for the production of a compound of the formula M H- Jwherein M is defined as above, with a haloacetylating agent selectedfrom the group consisting of bromoacetic anhydride, bromoacetylchloride, chloroacetic anhydride and chloroacetyl chloride, to obtain acompound of the formula wherein M is defined as above, and Z is selectedfrom the group consisting of chloro and bromo and reacting the latterwith a secondary amine to obtain the compounds of Formula C.

19. The process of claim 18 which includes the additional step ofreacting the compounds of Formula C with a pharmacologically acceptableacid to obtain the corre- 36v sponding pharmacologioally acceptable acidaddition salts of the compounds of Formula C.

20. The process of claim 19 which includes the additional step ofreacting the compounds of Formula C with a lower-alkyl halide to obtainthe corresponding quaternary ammonium salts of the compounds of FormulaC.

21. Process for the production of the S-aminoacetate quaternary ammoniumhalide derivatives of the com- .pounds of the formula wherein M is theremainder of the steroid moiety, which comprises reacting the compoundsof Formula B with a haloacetylating agent selected from the groupconsisting of bromoacetic anhydride, bromoacetyl chloride, chloroaceticanhydride and chloroacetyl chloride, to obtain a compound of the formulawherein M is defined as above and Z is selected from the groupconsisting of chloro and bromo, and reacting the compounds of Formula Dwith a tertiary amine to yield the corresponding 3-aminoacetatequaternary ammonium halides corresponding to Formula B.

References Cited by the Examiner UNITED STATES PATENTS 2,871,160 l/59Johnson et al. 167-77 2,883,401 4/59 Babcock et al 260397.45 3,029,2604/62 Mihina 260397.4 3,086,011 4/63 Hull 2602395 3,126,399 3/64 Sollman260397.4 3,157,679 11/64 Bork 260397.4

LEWIS GOTTS, Primary Examiner.

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF (A) THOSE HAVING THEFORMULAE: